Hydroxylation of Proteins

Hydroxylation is an oxidation reaction in which carbon–hydrogen (CH) bond oxidizes into carbon–hydroxyl (COH) bond. In organic chemistry, the hydroxylation reaction is mostly mediated by catalysts and heat. Most often the catalysts that mediates the hydroxylation reactions are metal ions. Hydroxylation of organic compounds converts hydrophobic molecules into hydrophilic molecules. Hydroxyl groups can form hydrogen bonds with water molecules and also with other organic functional groups such as amines, amides, and carboxyls. Hence, hydroxylated molecules become more water-soluble, which may impact the structure and function of organic molecules as well as the biomolecules. In biology, hydroxylation is mediated by enzymes called hydroxylases. Hydroxyproline residues are abundant in many proteins including collagen. Both 3-hydroxyproline and 4-hydroxyproline residues are present in collagen, an abundant matrix and structural protein. In addition, 5-hydroxylysine residues are also present in collagen and in other proteins. In collagen, the hydroxylysine residues are often glycosylated through the hydroxyl groups. The hydroxyproline and hydroxylysine residues play important roles in the water solubility as well as in the triple helical structure formation in collagen fibrils. In addition, hydroxyphenylalanine and hydroxytyrosine residues are also observed to exist in many endogenous proteins. Hydroxylation converts Phe residues into Tyr residues, which are important to control excess amounts of Phe residues in living organisms. Hydroxylation of Tyr at C-3 produces 3,4-dihydroxy phenylalanine (DOPA) that converts into dopamine and is a precursor to catecholamine hormones. Recombinant collagen and recombinant gelatin contain hydroxyproline and hydroxylysine residues that are important for their structural integrity and water solubility. Recombinant collagen is being used in tissue engineering and both recombinant collagen and recombinant gelatin are also used in drug delivery. Hydroxyl groups can be detected by both 1H and 13C NMR. High-resolution mass spectrometry is also a very useful tool to analyze hydroxylation. Since hydroxyl groups are hydrophilic, hydroxylation affects the chromatographic properties of molecules. Hence, hydroxylated molecules can be separated, characterized, and also quantitated by many separation methods including high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), capillary electrophoresis (CE), etc.